PROJECT SUMMARY Eukaryotic cells transport cargo between subcellular organelles, and to the plasma membrane for secretion, using small membrane-bound vesicles are carriers. The regulation of vesicular transport and membrane fusion processes are crucial for cellular morphology, growth, movement and secretion, including hormone release and neurotransmission. Many essential proteins are required for these processes, including the SNARE proteins and Sec1 that are involved in the membrane fusion process, the Rab and Rho GTPases, and a octameric tethering complex called the exocyst. Although the exocyst complex has been implicated in a number of different functions involved in recognition, tethering and quality control of SNARE assembly and fusion, none of these are well understood at the molecular level. We are using a multidisciplinary strategy of biochemical and biophysical techniques, combined with genetics and cell biological methods, in order to understand the molecular architecture and function of the exocyst complex. We study the exocyst proteins from the model organism Saccharomyces cerevisiae to take advantage of the wealth of genetic, cell biological and biochemical techniques available, but have expanded to exocyst from other organisms. Our studies aim to: 1) map the functional organization of the exocyst complex through biochemical studies in vitro and analyze mutants to test the function of the exocyst in vivo; (2) determine the 3D structure of the entire exocyst complex using electron microscopy, crystallography and molecular modeling; and (3) watch the exocyst tether vesicles at the single molecule level to analyze the requirements for tethering, and (4) dissect the role of the exocyst and Sec1 in SNARE complex assembly and membrane fusion. Because these proteins are conserved from yeast to human neurons, this research will advance our knowledge of how secretion and growth are regulated in all eukaryotic cells.